Mycobacterium tuberculosis RpfE crystal structure reveals a positively charged catalytic cleft

Abstract

Resuscitation promoting factor (Rpf) proteins, which hydrolyze the sugar chains in cell-wall peptidoglycan (PG), play key roles in prokaryotic cell elongation, division, and escape from dormancy to vegetative growth. Like other bacteria, Mycobacterium tuberculosis (Mtb) expresses multiple Rpfs, none of which is individually essential. This redundancy has left unclear the distinct functions of the different Rpfs. To explore the distinguishing characteristics of the five Mtb Rpfs, we determined the crystal structure of the RpfE catalytic domain. The protein adopts the characteristic Rpf fold, but the catalytic cleft is narrower compared to Mtb RpfB. Also in contrast to RpfB, in which the substrate-binding surfaces are negatively charged, the corresponding RpfE catalytic pocket and predicted peptide-binding sites are more positively charged at neutral pH. The complete reversal of the electrostatic potential of the substrate-binding site suggests that the different Rpfs function optimally at different pHs or most efficiently hydrolyze different micro-domains of PG. These studies provide insights into the molecular determinants of the evolution of functional specialization in Rpfs.

Keywords: electrostatic complementarity; enzyme specificity; lytic transglycosylase; peptidoglycan hydrolase; resuscitation promoting factor.

Figure 1

A: Ribbon representation of superimposed RpfE (blue) and RpfB (yellow) catalytic domain. The conserved, catalytic glutamate is shown in stick representation. B: RpfE and RpfB catalytic-cleft, Cα distance differences. C: Electrostatic potential surfaces (blue, > +5 kT and red, < −5 kT) of RpfE and RpfB calculated using the program Swiss-PdbViewer. RpfE shows positive potential around the predicted peptide-binding surfaces (arrows), while RpfB presents predicted peptide-binding surfaces (arrows) that are calculated to be negatively charged at neutral pH.

Figure 2

A: Superimposed RpfE (blue) and RpfB (yellow) catalytic domain bound to NAG₃. B: Superimposed RpfE (blue) and RpfB (yellow) residues in the catalytic cleft involved in binding NAG₃. All the residues are conserved between the two enzymes except for Gly350 substituted in RpfE by Arg163. C: RpfE surface with NAG₃ modeled in the catalytic cleft, based on a superposition with the structure of the RpfB-NAG₃ complex (PDB ID:4KPM). RpfE Arg163 overlaps with substrate, indicating that a conformational adjustment or a different ligand stereochemistry is necessary to accommodate the substrate in the catalytic cleft.

Figure 3

Sequence variation for RpfE and Mycobacterium leprae homologs. Conserved (blue) and variable residues (yellow) in RpfE and ML2151, ML2030, and ML0240, respectively, (A), (B), and (C). Sequence differences are prominent in the predicted peptide-binding groove to the right of the catalytic glutamate (dark blue) and the predicted carbohydrate-binding surface below the active-site pocket. D: Sequence variation between RpfE and the most similar protein in Micrococcus luteus (51% sequence identity) shows homologies in a more distantly related Rpf. E: Sequence aligment of Mtb RpfE and RpfB catalytic domain with M. leprae and M. luteus Rpfs.